NEIL3-ssDNA Structure Insights into Repairing Abasic Site Interstrand Crosslinks

Landová Barbora, Hušková Andrea, Bouřa Evžen, Šilhán Jan

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo náměstí 542/2, Praha 6, 16610

landova.barbora@uochb.cas.cz

Genomic DNA undergoes spontaneous base loss and deamination, followed by base removal. These are the leading causes for abasic site formation. The open-ring form of abasic ribose contains reactive aldehyde group that readily interacts with amines, occasionally leading to the formation of Abasic Site DNA interstrand crosslinks, known as Ap-ICL.

DNA crosslinks are blocking DNA replication and may result in cell cycle arrest, cell death, or cancer. Various repair processes have evolved to combat the deleterious effects. Replisome arrest triggers a ubiquitylation of replicative helicases recruiting the NEIL3 glycosylase for the repair of Ap-ICL. However, the molecular mechanisms of recognition and removal of Ap-ICL by this atypical DNA glycosylase remain elusive.

In this study, we present the crystal structure of the glycosylase domain of NEIL3 bound to single-stranded DNA (ssDNA) and a set of biophysical experiments aimed at providing a more comprehensive understanding of the process of Ap-ICL removal. Additionally, we characterize the resulting product of Ap-ICL removal as a 3’-phosphate. Our findings also demonstrate that the reduction of the Shiff-base of the Ap-ICL either impedes/does not affect its removal. Collectively, our data shed light on the molecular details of how NEIL3 glycosylase binds to and prefers ssDNA, rendering it a unique member of the Fpg/Nei family.

*For detailed experimental data see poster of Andrea Hušková